&lt;P&gt;In the last decade, the important role of small non-coding RNAs in regulation in all organisms have been recognized and begun to be studied. Our laboratory, in collaboration with others, have undertaken two completed global searches for non-coding RNAs in &lt;I&gt;E. coli&lt;/I&gt;, contributing significantly to the more than 80 regulatory RNAs that are now identified. A large number of these RNAs bind tightly to the RNA chaperone Hfq. We and others have shown that every RNA that binds tightly to Hfq acts by pairing with target mRNAs, regulating stability and translation of the mRNA, either positively or negatively. Our lab has studied a number of these small RNAs in detail. We have found that expression of each small RNA is regulated by different stress conditions, and that the small RNA plays an important role in adapting to stress.&lt;/P&gt;&lt;P&gt;One of the first studied small RNAs is RyhB. RyhB transcription is repressed by the Fur iron-dependent repressor, and the small RNA is therefore made in high quantities when intracellular iron is limiting. When it is made, it targets mRNAs that encode iron-binding proteins for degradation. Therefore, this small RNA, which is also found in &lt;I&gt;Vibrio&lt;/I., &lt;I&gt;Salmonella&lt;/I&gt;, &lt;I&gt;Klebsiella&lt;/I&gt;, and &lt;I&gt;Yersinia&lt;/I&gt;, reprograms iron use in the cells and may be an important component of virulence for some pathogens.&lt;/P&gt;&lt;P&gt;Two other small RNAs, now called OmrA and OmrB, regulate a number of outer membrane proteins;these small RNAs are made at high osmolarity as part of the OmpR/EnvZ regulon, previously known for its regulation of major outer membrane porins. In addition, OmrA and OmrB also regulate their own transcriptional regulators, providing a negative feedback loop that may be a frequent component of these regulatory systems. RybB, another Hfq-binding RNA, is dependent on an alternative sigma factor, Sigma E, for transcription and down-regulates outer membrane proteins. These RNAs are characteristic of a growing family of regulatory RNAs that regulate the cell surface, possibly important during infection. An additional outer membrane protein, OmpX, has now been shown to be regulated by CyaR, also an Hfq-binding RNA that is positively regulated by cyclic AMP and CRP. In addition to OmpX, believed to be important for cell adhesion, CyaR down-regulates the synthesis of other proteins, including LuxS, the synthase for a quorum-sensing molecule believed to work for a broad range of species. It seems possible that CyaR helps the cell down-regulate functions important in escape from biofilms under poor nutrient conditions (low glucose).&lt;/P&gt;&lt;P&gt;Consistent with the idea that all major regulatory systems may have small RNA components, another Hfq-binding RNA, named MgrR, is regulated by PhoP and PhoQ, a two-component system important for Salmonella virulence. PhoP and PhoQ activate synthesis of the RNA under low Magnesium conditions;the small RNA inactivates an enzyme for modification of the cell surface lipopolysaccharide, affecting the cells sensitivity to antimicrobial peptides such as polymyxin. This is the first example of regulation of an LPS modifying enzyme by sRNAs.&lt;/P&gt;&lt;P&gt;Previous studies had demonstrated the roles of two small RNAs, DsrA and RprA, in positively regulating translation of the stress sigma factor RpoS. More recently, we have shown that RprA also has a number of other mRNA targets, which are negatively regulated;these targets differ from those for DsrA. The new RprA targets expand the likely role of RprA and its regulators, RcsC, RcsD, and RcsB, in controlling biofilm formation by this bacteria. Studies on the mechanism of action of DsrA and RprA suggest that they increase both the stability and translation of &lt;I&gt;rpoS&lt;/I&gt;mRNA, protecting it from degradation by RNAse E.&lt;/P&gt;&lt;P&gt;In other experiments, we have chosen genes with no known small RNA regulators, created translational fusions to them, and used genetic screens to identify sRNA translational regulators. In particular, we found that the &lt;I&gt;dpiAB&lt;/I&gt;genes are regulated by a previously uncharacterized Hfq-binding RNA, RybC. Because DpiAB are involved in helping cells survive in the presence of low levels of antibiotic, it is possible the small RNA is involved in this response. The approach used to study &lt;I&gt;dpiAB&lt;/I&gt;has been developed into a very flexible set of strains to allow the rapid analysis of many targets, already being used for many lab projects. Because we believe that essentially all of the Hfq-dependent sRNAs have now been identified, a library of expression plasmids for each of these sRNAs has been made and now can be used to rapidly screen for regulation of a given target fusion. Extending the work on translational regulation of RpoS and other sigma factors, a third sRNA, RyhA, was found to strongly stimulate RpoS translation by direct pairing. RyhA is regulated in response to switches from aerobic to anaerobic growth. A set of other sRNAs have been found to modestly stimulate RpoS expression, possibly by effects on the three known direct regulators;yet others negatively regulate RpoS, possibly by competing for the Hfq protein.&lt;/P&gt;&lt;P&gt;In order to determine if factors other than Hfq are necessary for the action of these sRNAs, a genetic selection was developed to select for failure of two sRNAs to act. Among the mutations isolated were changes in conserved and essential amino acids in hfq and loss of function mutations in pnp, encoding polynucleotide phosphorylase, and in pcnB, encoding polyA polymerase. pnp mutations lead to decreased levels of some sRNAs, and this decreased accumulation may be sufficient to explain their failure to act. However, how lack of pnp leads to this phenotype remains to be investigated.&lt;/P&gt;&lt;P&gt;Our work, combined with work from other labs on this family of regulators, suggests that a large number of genes in bacteria will be subject to this post-transcriptional regulation.&lt;/P&gt;&lt;P&gt;Tiling arrays allow a detailed examination of the RNA transcripts in the cell. In collaboration with Dr. G. Storz, we are using an &lt;I&gt;E. coli&lt;/I&gt;tiling array to examine whether any additional small RNAs are present in E. coli and to define possible mRNA target RNAs.&lt;/P&gt;&lt;/P&gt;In addition, in collaborative work, small RNAs in B. anthracis are being identified and characterized, as is the role of the multiple Hfq species in that organism. This provides a useful comparison to the E. coli work.&lt;/P&gt;